MULTICOLOR PREPARATIONS FOR IN-SITU HYBRIDIZATION USING PRECIPITATINGENZYME CYTOCHEMISTRY IN COMBINATION WITH REFLECTION CONTRACT MICROSCOPY

We have further developed a method for the detection of different enzy me cytochemical reaction products by means of reflection contrast micr oscopy (RCM). By embedding these enzyme precipitates in a protein matr ix, we were able to prevent the reaction products from dissolving in i mmersion oil, which is required for RCM analysis. The applicability of the RCM procedure is, therefore, extended to a range of cytochemical enzyme precipitation methods, which normally result in oil soluble rea ction products. To test their usefulness, these enzyme precipitates ha ve been used in single- as well as double-label in situ hybridization (ISH) procedures to visualize a number of DNA target sequences by seve ral different reflection colours, i.e. white, yellow and red. Three re petitive DNA probes for the (sub)centromeric regions of chromosomes 1, 7 and 17, as well as a repetitive DNA probe for the telomeric region of chromosome 1, and two cosmid DNA probes (40 kb each) for both arms of chromosome 11 could bc detected with high efficiency in both interp hase and metaphase preparations. Moreover the enzyme precipitates were shown to be stable upon exposure to excitation light or upon storage. It may be concluded that these findings render RCM a sensitive method for the visualization of multiple targets in biological specimens.